Hypothalamic transcriptional expression of the kisspeptin system and sex steroid receptors differs among polycystic ovary syndrome rat models with different endocrine phenotypes

OBJECTIVES: Polycystic ovary syndrome is a heterogeneous endocrine disorder that affects reproductive-age women. The mechanisms underlying the endocrine heterogeneity and neuroendocrinology of polycystic ovary syndrome are still unclear. In this study, we investigated the expression of the kisspeptin system and gonadotropin-releasing hormone pulse regulators in the hypothalamus as well as factors related to luteinizing hormone secretion in the pituitary of polycystic ovary syndrome rat models induced by testosterone or estradiol. METHODS: A single injection of testosterone propionate (1.25 mg) (n=10) or estradiol benzoate (0.5 mg) (n=10) was administered to female rats at 2 days of age to induce experimental polycystic ovary syndrome. Controls were injected with a vehicle (n=10). Animals were euthanized at 90-94 days of age, and the hypothalamus and pituitary gland were used for gene expression analysis. RESULTS: Rats exposed to testosterone exhibited increased transcriptional expression of the androgen receptor and estrogen receptor-β and reduced expression of kisspeptin in the hypothalamus. However, rats exposed to estradiol did not show any significant changes in hormone levels relative to controls but exhibited hypothalamic downregulation of kisspeptin, tachykinin 3 and estrogen receptor-α genes and upregulation of the gene that encodes the kisspeptin receptor. CONCLUSIONS: Testosterone- and estradiol-exposed rats with different endocrine phenotypes showed differential transcriptional expression of members of the kisspeptin system and sex steroid receptors in the hypothalamus. These differences might account for the different endocrine phenotypes found in testosterone- and estradiol-induced polycystic ovary syndrome rats.

Musk gland seasonal development and musk secretion are regulated by the testis in muskrat (Ondatra zibethicus)

BACKGROUND: The muskrat is a seasonal breeder. Males secrete musk to attract females during the breeding season. The testosterone binding to the androgen receptor (AR) in musk glands of muskrat may play an important role conducting the musk secretion process. METHODS: The musk gland, testis and blood samples of musk rats are collected in both breeding and non-breeding seasons. Some part of the samples are kept in liquid nitrogen for transcriptome analysis and Western blotting test. Some part of the samples are kept in 70% alcohol for histology experiment, blood samples are kept at -20 °C for the serum testosterone measurement experiment. RESULTS: This study demonstrates that the quantity of secreted musk, the volume of the musk glands, the diameter of the gland cells and AR expression are all higher during the breeding season than at other times (p < 0.01). StAR, P450scc and 3ß-HSD expression in the Leydig cells of the testis were also higher during this season, as was serum testosterone. AR was also observed in the gland cells of two other musk-secreting animals, the musk deer and small Indian civet, in their musk glands. These results suggest that the testes and musk glands co-develop seasonally. CONCLUSION: The musk glands' seasonal development and musk secretion are regulated by the testes, and testosterone plays an important role in the seasonal development of musk glands.

Expression of procaspase 3 and activated caspase 3 and its relevance in hormone-responsive gallbladder carcinoma chemotherapy

BACKGROUND/AIMS: The higher incidence of gallbladder cancer (GBC) in females has been accredited to the involvement of hormones. The clinical implications of sex hormone receptors in GBC are well established. Cysteine proteases (such as caspase-3-9, etc.) are known to play a central role in the apoptotic pathway. Of these, the downstream enzyme caspase-3 is often activated in the apoptotic pathway. The aim of this work was to examine the status of apoptosis (which directly correlated with the level of active caspase-3) in hormone-responsive GBC. METHODS: We used 10 androgen receptor (AR)-positive, 14 estrogen receptor (ER)-positive, 12 HER/neu-positive, eight triple positive, and 10 triple negative malignant GBC human tissue samples. We isolated the total cellular protein from tumor tissues and carried out Western blotting using antipro-caspase-3 and anti-activated caspase-3 antibodies. RESULTS: ER and HER/neu-positive GBC exhibited high caspase-3 activity and low procaspase-3 activity, whereas AR-positive GBC showed no significant level of apoptosis. We also evaluated the apoptosis status of triple positive GBC and triple negative GBC, and found significant apoptosis in triple positive GBC. CONCLUSIONS: The results indicate that ER and HER/neu-positive GBCs had active apoptosis, whereas AR-positive GBC was highly resistant to apoptosis.

The effect of microgravity on tissue structure and function of rat testis

To explore whether an environment of weightlessness will cause damage to the reproductive system of animals, we used the tail-suspension model to simulate microgravity, and investigated the effect of microgravity on the tissue structure and function of the testis in sexually mature male rats. Forty-eight male Wistar rats weighing 200-250 g were randomly assigned to three groups (N = 16 each): control, tail traction, and tail suspension. After the rats were suspended for 7 or 14 days, morphological changes of testis were evaluated by histological and electron microscopic methods. The expression of HSP70, bax/bcl-2 and AR (androgen receptor) in testis was measured by immunohistochemistry. Obvious pathological lesions were present in the testis after the rats were suspended for 7 or 14 days. We detected overexpression of HSP70 and an increase of apoptotic cells, which may have contributed to the injury to the testis. The expression of AR, as an effector molecule in the testis, was significantly decreased in the suspended groups compared to control (P < 0.01). We also observed that, with a longer time of suspension, the aforementioned pathological damage became more serious and some pathological injury to the testis was irreversible. The results demonstrated that a short- or medium-term microgravity environment could lead to severe irreversible damage to the structure of rat testis.

Absolute measurement of androgen receptor mRNA in peripheral blood mononuclear, preputial skin and urethral mucosa cells of control individuals with phimosis using qRT-PCR / Valor absoluto do mRNA do receptor androgênico em células mononucleares do sangue periférico e células da pele do prepúcio e mucosa uretral em indivíduos controle com fimose, determinado por meio de qRT-PCR

INTRODUCTION: Androgen actions are exerted upon the androgen receptor (AR), and complete genital virilization of normal 46,XY individuals depends on adequate function and expression of the AR gene in a tissue-specific manner. OBJECTIVE: Standardization of normal ARmRNA in androgen-sensitive tissues. MATERIALS AND METHODS: In this study, we determined the quantitative amounts of ARmRNA in peripheral blood mononuclear, urethral mucosa and preputial skin cells of control subjects with phimosis by using RT-PCR. RESULTS: The mean (SD) values of AR expression in blood, urethra and prepuce were: 0.01 (0.01); 0.43 (0.32); 0.31 (0.36), respectively. CONCLUSION: The AR expression is low in blood and equivalent in urethral mucosa and preputial skin, which may be useful in the diagnosis of individuals with abnormal external genitalia.

INTRODUÇÃO: As ações androgênicas são exercidas por meio do receptor androgênico (AR), e a completa virilização genital de indivíduos 46,XY normais depende de adequada expressão do gene AR de forma tecido específica. OBJETIVO: Padronizar valores normais de ARmRNA em tecidos sensíveis aos andrógenos. MATERIAIS E MÉTODOS: Neste estudo, determinamos as quantidades de ARmRNA em células mononucleares do sangue periférico e em células da mucosa uretral e pele do prepúcio de indivíduos controles com fimose, utilizando RT-PCR. RESULTADOS: A média (dp) dos valores de expressão do AR em sangue, uretra e prepúcio foram: 0,01 (0,01); 0,43 (0,32); 0,31 (0,36), respectivamente. CONCLUSÃO: A expressão do AR é baixa em sangue periférico e equivalente em mucosa uretral e pele prepucial, sendo sua quantificação útil no diagnóstico de indivíduos com alterações da genitália externa.

Stem cells and androgen receptor in human periapical periodontitis / Células madre y receptor de andrógenos en periodontitis periapical humana

Activation of macrophages in periapical granulomas occurs through the presence of cytokines, endotoxin and other genetic and epigenetic factors, allowing the initiation of inflammation and bone resorption. The present study aims to analyze the presence of CD133 protein (marker of stem cells) and the AR (androgen receptor) protein in biopsies of human odontogenic periapical granuloma. Biopsies from 14 adult male patients with diagnosis of periapical granuloma included in paraffin blocks were processed histologically to obtain 5-um thick sections. Protein presence was detected and analyzed by immunohistochemistry of CD133 and AR. The quantification considered the number of positive cells in 0.17 mm2 random areas under the microscope using a 1000X objective. Both CD133 and AR proteins are expressed abundantly in cells in pathological periapical granulomas tissue. The number of cells expressing CD133 and AR shows a wide variation coefficient, so its variation is a particular feature for each individual. We concluded that in human odontogenic periapical granuloma there are abundant stem cells and cells expressing AR that may be important for the pathogenic inflammatory process.

La activación de los macrófagos en los granulomas periapicales humanos se producen a través de la presencia de citoquinas, endotoxinas y otros factores genéticos y epigenéticos que permiten la iniciación de la inflamación y la reabsorción ósea. El presente estudio pretende analizar la presencia de proteína CD133 (marcador de células madre) y de la proteína RA (receptor de andrógenos) en las biopsias de granulomas periapicales odontogénicos humanos. Las biopsias de 14 pacientes varones adultos con diagnóstico de granuloma periapical fueron incluidos en bloques de parafina y se procesaron histológicamente para obtener secciones de 5 micras de espesor. La presencia de CD133 y RA fueron detectadas y analizadas por inmunohistoquímica. La cuantificación se realizó considerando el número de células positivas en áreas al azar de 0,17mm2, utilizando microscopio con objetivo de 1000X. Ambas proteínas, CD133 y RA se expresan en abundancia en las células del tejido patológico con granuloma periapical. El número de células que expresan CD133 y RA presentan un amplio coeficiente de variación, por lo que su variación es una característica particular de cada individuo. Se concluye que en granuloma periapical odontogénico humano se expresan abundantes células madre y proteínas receptoras de andrógenos, antecedentes que pueden sermuy importantes en la expresión y diagnosis de los procesos patológicos inflamatorios.

Parallel determination of NeuroD1, Chromogranin-A, KI67 and androgen receptor expression in surgically treated prostate cancers

PURPOSE: Neuroendocrine differentiation is a hallmark of prostate cancer. The aim of our study was the detection of the parallel expression of neuroendocrine related markers using a prostate tissue microarray (TMA). MATERIALS AND METHODS: Our study was aimed at detecting the parallel expression of NeuroD1, Chromogranin-A (ChrA), Androgen Receptor (AR) and Ki-67 by immunohistochemistry on prostate cancer tissue microarray. The data was analyzed using SAS version 8.2 (SAS Inc, Cary, NC). The relationships between NeuroD1, ChrA and AR expressions and patients' characteristics were investigated by multivariate logistic regression analysis. Progression and Overall Survival (OS) distributions were calculated using Kaplan-Meier method. RESULTS: Tissue reactivity for NeuroD1, ChrA and AR concerned 73 percent, 49 percent and 77 percent of the available cases, respectively. Regarding overall survival, there were 87 deaths and 295 patients alive/censored (6 years of median follow-up). Seventy-seven disease progressions occurred at the median follow-up 5.4y. A significant correlation between NeuroD1, ChrA and AR expression was observed (p < 0.001 and p < 0.03, respectively). Additionally, ChrA was strongly associated in multivariate analysis to Gleason score and Ki67 expression (p < 0.009 and p < 0.0052, respectively). Survival analysis showed no association between markers neither for overall nor for cancer-specific survival. CONCLUSIONS: The results highlight that NeuroD1, Chromogranin-A and Androgen Receptor are strongly associated, however their expression does not correlate with overall survival or disease progression.

Immunohistochemical study of androgen, estrogen and progesterone receptors in salivary gland tumors

The aim of this work was to study the immunohistochemical expression of androgen receptor, estrogen receptor and progesterone receptor in pleomorphic adenomas, Warthin's tumors, mucoepidermoid carcinomas and adenoid cystic carcinomas of salivary glands. A total of 41 pleomorphic adenomas, 30 Warthin's tumors, 30 mucoepidermoid carcinomas and 30 adenoid cystic carcinomas were analyzed, and the immunohistochemical expression of these hormone receptors were assessed. It was observed that all cases were negative for estrogen and progesterone receptors. Androgen receptor was positive in 2 cases each of pleomorphic adenoma, mucoepidermoid carcinoma and adenoid cystic carcinoma. In conclusion, the results do not support a role of estrogen and progesterone in the tumorigenesis of pleomorphic adenomas, Warthin's tumors, mucoepidermoid carcinomas and adenoid cystic carcinomas. However, androgen receptors can play a role in a small set of salivary gland tumors, and this would deserve further studies.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14 percent (5/36), or 55 percent (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

Androgen & vitamin D nuclear receptor expression in archival breast tumour samples.

BACKGROUND & OBJECTIVE: Breast tumour cells have receptors for androgen and vitamin D and their clinical significance is not completely understood. Therefore, the present study was undertaken to analyze androgen and vitamin D receptor levels in human primary infiltrating ductal breast carcinomas (IDC) and benign breast tumour archival samples and to find out their correlation, if any, with the clinical findings. METHODS: Paraffin blocks of benign and malignant breast tumours were sectioned, deparaffinized, and nuclei released by pepsin digestion. After antigen retrieval, nuclei were stained with primary antibodies for androgen or vitamin D receptors and secondary fluorescein isothiocyanate (FITC) labeled antibodies and propidium iodide respectively, to quantitative receptor expression and DNA content by flow cytometry. RESULTS: Androgen receptor positive nuclei ranged from 16-66 per cent in the IDC tumours as compared to 36-67 per cent in the benign tumours. Based on flow cytometric comparison of AR expression in AR positive and negative cell lines established earlier, 24 of 28 tumours from postmenopausal women were AR positive compared to all benign tumours and 32 of 33 tumours from pre-menopausal patients. Vitamin D receptor positive nuclei ranged from 14-89 and 2-75 per cent in IDC and benign tumours, respectively. All pre- or post-menopausal tumours were VDR positive as compared to 10 of 15 benign tumours that were VDR positive. No correlation was seen between nuclear androgen and vitamin D receptor expression of the IDC or benign tumours. There was a positive correlation between per cent of receptor positive nuclei and antigen density as measured by ratio of the mean log fluorescence channel value (MFC). No statistically significant correlation was found between nuclear receptor expression (per cent positive nuclei or antigen density) with that of tumour stage, lymph node status, tumour grade, patient age or menopausal status. INTERPRETATION & CONCLUSION: There was no significant correlation between androgen or vitamin D receptor expression and clinical findings. The expression of AR and VDR and the antigen density in the nuclei of the archival breast tumour samples were highly variable because of the tumour heterogeneity. Future studies with fresh biopsy samples of tumour on AR and VDR levels and their up- or down-regulation may be useful while stratifying the patients for hormonal therapy.

Câncer da próstata: recurso diagnósticos atuais / Prostate carcinoma: current diagnostic strategy

Câncer da próstata é a segunda causa de morte por câncer na populaçäo masculina ocidental. Apesar do progresso no tratamento dos estádios avançados, näo há dúvidas de que a única possibilidade de reduçäo da mortalidade é o diagnóstico precoce, quando a doença ainda está localizada. Neste estudo, objetivamos rever os recursos atuais disponíveis para o diagnóstico do carcinoma prostático. Antígeno prostático específico (PSA) é importante marcador tumoral e tem demonstrado ser efetivo na detecçäo, na monitorizaçäo terapêutica e na identificaçäo de recorrência. Mesmo assim, métodos alternativos têm sido propostos, como a relaçäo entre o PSA livre e o total, a densidade e a velocidade do PSA, os quais podem melhorar a sensibilidade e a especificiddade diagnósticas. Métodos de imagem incluem ultra-som transretal, tomografia computadorizada, ressonância magnética e mapeamento ósseo. O ultra-som é o melhor método para direcionar a realizaçäo de biópsias e, juntamente com a ressonância magnética, pode ser útil no estadiamento locorregional. Tanto a tomografia como a ressonância podem ser utilizadas para a identificaçäo de comprometimento linfonodal. O mapeamento ósseo é o melhor método para diagnóstico de metástases à distância

Cuantificación de receptores androgénicos como factor pronóstico en pacientes con cáncer de próstata / Quantitative determination of tissue androgenic receptors as a prognostic factor in patients who have prostatic carcinoma

El cáncer de próstata es el tumor maligno más común en la población masculina mayor de 50 años y está considerado como la segunda causa de muerte por cáncer. El 70 por ciento de los pacientes al momento de su diagnóstico presentan enfermedad avanzada por lo que la gran mayoría de ellos recibirán tratamiento hormonal, predecir qué pacientes responderán a éste es el objetivo de este estudio. De enero de 1991 a junio de 1994 se atendieron 40 pacientes con diagnóstico de cáncer de próstata estadio D2, con un seguimiento mínima de dos años. Se reconfirmó la gradación de Gleason y de sus bloques de parafina se realizó la cuantificación de los receptores por técnica de inmunoeroxidas. Presentaron buena respuesta 25 casos (62.5 por ciento) es decir más de dos años de sobrevida libre de actividad tumoral y mala respuesta 15 (37.5 por ciento) quienes presentaron actividad tumoral antes de los dos años o que presentaron nula respuesta al tratamiento. La edad de nuestra población fue de 71 (ñ 19) años, el antígeno prostático específica presentó un rango variable entre 11.2 y 900 ng/ml con una mediana de 41.6 ng/ml. En la gran mayoría de nuestros pacientes su gradación de Gleason fue de 2, 3 y 4. La cuantificación de receptores en los casos con buena respuesta de 121.5 /ñ 21.3). La calidad de la respuesta al tratamiento fue directamente proporcional al número de receptores (p<0.005), de donde concluimos que la determinación de dichos receptores sirve como factor pronóstico en pacientes con cáncer de próstata y que aún más puede predecir cuáles pacientes serán buenos candidatos al tratamiento hormonal

Immunoreactivity of androgen receptor protein in sexually dimorphic spinal motonucleus in neonatal male rats

The spinal motonucleus of the genitofemoral nerve regulating scrotal temperature can also be related to prenatal and neonatal testicular descent by gubernacular change in rats, and a sexually dimorphic-like bulbocavernosus/dorsolateral motonucleus. There is a hypothesis that neonatal androgen affects these motonuclei, and induces development of sexual organs through neural stimulation. Until now, the accumulation of isotope-labelled androgen and the immuno-reactivity of androgen receptor protein in each sexually-dimorphic spinal motonucleus have been revealed in adult rats but they have not been established in rats during neonatal periods. To investigate the presence of the androgen receptor in spinal sexually-dimorphic motonuclei in the neonatal period, we evaluated the androgen receptor immunoreactivity of these motonuclei. In Sprague-Dawley male rats, the lumbar spinal cords were resected at postnatal days 3, 10 and 30, and stained immunohistochemically using polyclonal antibody of androgen receptor protein. The immunoreactivity of androgen receptor protein was observed in the cells of the genitofemoral motonucleus from the 13th thoracic to the 2nd lumbar spinal cord and the bulbocavernosus/dorsolateral motonucleus was observed from the 4th to 5th lumbar spinal cord in all age groups. The proportional areas of both motonuclei at days 3 and 10 on cross-section were larger than at day 30. The motonuclei at days 3 and 10 were similar in all age groups. With the above results, the presence of androgen receptor protein was confirmed in the genitofemoral and bulbocavernosus/dorsolateral motonucleus from neonate to day 30. The larger proportional area of these motonuclei in neonates may indicate an active role for these motonuclei during the neonatal period. Although the immunoreactivity does not directly imply the presence of a functional receptor, neonatal androgen could be responsible for the development of sexual organs through the spinal motonucleus.

Androgen binding site in macrophages

The presence of androgen binding sites [five alpha dihydrotestosterone [5%-DHT]] on NZB/BALBc mouse peritoneal macrophages [M phi] was identified. Cells were incubated with different concentrations of [3H]-5 alpha-DHT in the presence or absence of a 100 fold excess of unlabelled 5 alpha-DHT to correct for nonspecific binding. Labelled cells were separated from unbound steroid by immunomagnetic beads coated with rat anti-mouse M phi antibody [rat anti-mouse F4/80 Ag]. The binding identified in the mouse M phi was highly selective towards androgenic compounds. Little competition was seen from 17 beta oestradiol [17beta E2] or the synthetic glucocorticoid triamcinolone acetonide. Binding data derived from Scatchard analysis showed the presence of a high affinity androgen binding species. The dissociation constant [Kd] value for the receptor was calculated to be 4.7x10[-9]M. The number of receptors per cell was 2950

Steroid receptors in meningiomas

Dados epidemiológicos sugerem uma associaçäo entre meningiomas e hormônios sexuais. Há preponderância da incidência da incidência destes tumores em mulheres, sendo diagnosticados mais frequentemente entre 35 e 55 anos. Progressäo tumoral pode ocorrer durante a gravidez e a associaçäo entre meningiomas e câncer de mama é estatisticamente significativa. O efeito benéfico positivo dos glicocorticóides no tratamento clínico de tumores intracranianos está bem documentado, embora este efeito seja relacionado com o edema cerebral e näo com proliferaçäo tumoral. A presença de receptores intracelulares específicos parece ser condiçäo necessaria embora näo suficiente para a expressäo dos efeitos celulares destes esteróides. O objetivo do nosso trabalho foi a determinaçäo de receptores de estrógeno (ER), progesterona (PR), andrógeno (AR) e glicocorticóide (GR) em 10 meningiomas através da metodologia do carväo-dextrana. Neste estudo verificamos que somente 20% dos meningiomas contém receptores de estrógeno e as concentraçöes determinadas säo baixas. Receptores de progesterona estäo presentes em 90% dos tumores, em alta concentraçäo (média + ou - dp = 60 + ou - 38 fMol/mg prot). Cerca de 70% dos meningiomas contém níveis intermediários de AR e GR. Testes de competiçäo demonstram que todos os receptores säo específicos. Incidência e concentraçäo de todos os receptores säo maiores em pacientes do sexo feminino. A ocorrência de receptores de progesterona, andrógeno e glicocorticóide pode indicar a possível utilidade de manipulaçäo endócrinas em meningiomas näo operáveis